Project Summary: Chronic hepatitis C virus (HCV) infection affects about 3 percent of the world's population and HCV cirrhosis is now the leading indication for liver transplantation in the United States. The best current therapy for chronic HCV infection (combination peginterferon and ribavirin) leads to sustained virologic responses in only about half of all treated individuals. However, there are subgroups that have much lower rates of treatment response, such as African Americans and people with HIV coinfection. There is therefore a great need for novel HCV therapies. Small molecule inhibitors of HCV proteins are promising, but it is clear that viral resistance develops quickly to such agents. We propose to identify host genes that serve as viral replication cofactors, with the rationale that cellular cofactors may be targets for HCV therapy. For example, cyclophilin B is a host protein that has been shown to be involved in HCV replication, and small molecule inhibitors of cyclophilin B are potent inhibitors of HCV replication both in vitro and in vivo. In this application, we will screen a whole human genome siRNA library against a HCV replicon cell line that encodes a luciferase reporter gene. siRNA downregulation of essential cellular cofactors is expected to block HCV replication and luciferase activity in this system. Preliminary studies show that high-throughput siRNA transfection of a HCV replicon cell line is feasible. We discuss methods of hit selection, as well as secondary screening steps designed to minimize the number of false positive results. Public Health Relevance Hepatitis C virus infection can cause serious liver disease, including cirrhosis and liver cancer. The best current therapy for HCV only works in about half of all treated people, and also can cause serious side effects. This project hopes to discover the human proteins that the virus needs to reproduce inside cells, as these might turn out to be possible targets for new HCV therapies. [unreadable] [unreadable] [unreadable]